The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways.Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the in vitro kinetics of ubiquitination. FRET is established between binding of fluorophorelabeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.

The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for the conjugation of Ub to cellular proteins, thereby controlling biological activities.Due to the numerous enzymes and processes involved in ubiquitination, studies on the molecular mechanisms have been challenging. We here report a novel FRET-based assay to study the kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model enzymes, we demonstrate that ubiquitination rates can be measured by FUSS using the rate of change in the FRET efficiency over time. Treatment with USP21, a deubiquitinase, removes ubiquitination on CHIP and leads to increased FRET efficiency, confirming the reversibility of the assay. Incubation with tau, a substrate of CHIP, increases the overall ubiquitination activity. We further use this assay to measure the relationship between increasing E1, E2 or E3 concentrations and ubiquitination rates, revealing that Uba1 activity is the ratelimiting step. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus potentially compatible with any E1-E2-E3 combinations. (214 words)